The cellular and molecular processes involved in the response of neoplastic epithelial cells to radiation are largely unknown. While in some cell types, particularly of reticuloendothelial origin, death following irradiation is preceded by apoptotic changes, apoptosis plays little or no role in the death of epithelial neoplastic cells following radiation (Bristow et al., 1996, Radiotherapy & Oncol. 40:197-223; Brown et al., 1999, Cancer Res. 59:1391-1399; Finkel, 1999, Science 286:2256-2258). Epithelial cells do not undergo apoptosis following irradiation and are therefore likely to respond with a different sequence of programmed cytoplasmic and nuclear events.
It has been proposed that there are two fundamental mechanisms of cell death (Schwartz et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:980-984; Zakeri et al., 1995, Cell Death and Differentiation 2:87-96), referred to as Type I and Type II programmed cell death. Type I programmed cell death, also known as apoptosis, is mediated by a cascade of cysteine aspartases (caspases) and factors released by mitochondria (Reed, 1999, J. Clin. Oncol. 17:2941-2953) and has typical morphological and biochemical characteristics, such as chromatin margination and condensation, early nuclear collapse and nucleosomal ladder formation (Zakeri et al., 1995, Cell Death and Differentiation 2:87-96). In contrast, Type II programmed cell death is marked morphologically by increased autophagy and early destruction of the cytoplasm that either occurs without nuclear collapse or precedes it. Type II programmed cell death has been documented mainly in Lepidoptera during metamorphosis and during involution of rat mammary gland (Schwartz et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:980-984; Zakeri et al., 1995, Cell Death and Differentiation 2:87-96), but has rarely been associated with stress-inducing stimuli (Bursch et al., 1996, Carcinogenesis 17:1595-1607; Jia et al., 1997, Br. J. Hematol. 98:673-685). Unfortunately, methods for quantification of Type II programmed cell death are lacking, and the molecular mechanisms that regulate it have not been defined.
There are four major classes of ATPases, (i) P-ATPases which use the energy released by ATP hydrolysis to translocate cations across membranes and form a phosphorylated intermediate, i.e., Na+/K+ ATPase; (ii) F-ATPase which utilize energy stored in electrochemical gradient to synthesize ATP, i.e., mitochondrial ATP synthase; (iii) ATP binding cassette (ABC) transporters which mediate efflux of a variety of solutes by hydrolyzing ATP; and (iv) V-ATPase which hydrolyze ATP to create a proton gradient (Lodish, et al., 2000 Molecular Cell buiology ed. S. Tenney Publisher W. H. Freeman and Company).
Vacuolar proton ATPase (“V-H+-ATPase”) belongs to a special class of ATPases (V-type) composed of two multi-subunit sectors. The two sectors include a peripheral detachable sector (V1) containing the catalytic ATPase domain and an integral membrane sector (V0) responsible for proton translocation across membranes. The V-H+-ATPase is responsible for acidification of cellular organelles such as endosomes, lysosomes, secretory granules and in some cells the trans-golgi cisternae. Studies with specific inhibitors of the enzyme have shown that maintaining low pH in the acidic compartments is essential for proper vesicular traffic and protein sorting within the cells.
In addition to their role in acidic subcellular organelles, V-ATPases are also found in the plasma membrane of some cancer cells and of specialized cells that specialize in proton secretion such as macrophages, epididymal cells, osteoclasts, renal intercalcalated cells and epididymal cells. In these cells V-ATPases contribute to pH homeostasis, bone resorption and renal acidification (Drose et al,1997, J. Exp. Biology 200:1-8; Gagliardi et al., 1999, Current Medicinal Chemistry 6:1197-1212; Forgac et al 1999).
Specific inhibitors of V-ATPase belong to two major chemical groups the plecomacrolydes (Drose and Altendorf 1997, J. Exp. Biology 200:1-8; Gagliardi et al. 1999, Current Medicinal Chemistry 6:1197-1212) and the benzolactone enamides (Boyd and Farina et al., 2001, J. Pharmacology and Experimental Therapeutics 297:114-120). The most commonly used inhibitors bafilomycins and concanamycins—belong to the first group while the newly discovered salicylihalamide A, lobatamides A-F and oximidines I and II belong to the second group. Bafilomycins and concanamycins are closely related macrolides that possess a six-member hemiacetal ring connected via C3 spacer to a 16 (bafilomycins) or an 18 (concanamycins) member macrocyclic lactone ring. In concanamycins the hemiacetal ring is glycosylated (Drose and Altendorf, 1997, J. Exp. Biology 200:1-8).
Concanamycins and bafilomycins inhibit V-ATPase at nanomolar concentrations. However, they do not affect the activity of F-ATPases and inhibit P-ATPases and ABC transporters only at micromolar concentrations. Therefore, inhibition of an H+-ATPase by nanomolar concentrations of bafilomycins or concanamycins is taken as a criterion for the classification of that enzyme as a V-H+-ATPase (Drose and Altendorf, 1997, J. Exp. Biology 200:1-8). While the IC50 of bafilomycins and concanamycins in in vitro assays is lower than 1 nM, the concentrations needed to affect complete inhibition of acridine orange accumulation by lysosomal compartments in whole cells can reach a few hundreds nanomolar (Drose and Altendorf, 1997, J. Exp. Biology 200:1-8). It has been suggested that because bafilomycins and concanamycins are lipid soluble they may be sequestered in different organelles and thus higher concentrations are required to achieve an effective amount at their targets (Drose and Altendorf, 1997, J. Exp. Biology 200: 1-8).
Bafilomycins and concanamycins are thought to exert their inhibitory effect on V-ATPases by interacting with subunits within its integral membrane complex—Vo, and there is evidence suggesting that the proton channel-forming subunit c and a 116 Kd subunit interact directly with the inhibitors (Drose and Altendorf, 1997, J. Exp. Biology 200:1-8).
Demonstration of V-H+-ATPase-dependent acidification of cellular organelles as well as its involvement in different cellular processes has been achieved by employing the specific inhibitor bafilomycin A1 (Gagliardi, S., et al., 1999, Current Medicinal Chemistry 6:1197-1212). When used at low concentrations (nM range), bafilomycin A1 inhibits V-H+-ATPase activity without affecting the activity of either F or P-type ATPases. However, when bafilomycin A1 is used at the μM range F and P-type ATPases activities are affected.
Several studies reported that long-term incubation of cultured cells with bafilomycin A1 resulted in cell death (Drose and Altendorf 1997; Gagliardi, Rees et al. 1999). Additionally, a concomitant inhibition of V-H+-ATPase with bafilomycin A1, and of Na+/H+ antiporter with 5-(N-ethyl-N-isopropyl)-amiloride in human breast cancer cells results in decreased cytoplasmic pH and increased DNA degradation (Thangara, M., Cancer Research 59:1649-1654). However, Manabe et al. (Manabe et al., 1993 J. Cell Physiol 157:445-452) claim that removal of the compound following short-term incubation resulted in resumption of cell growth. Because bafilomycin A1 binds tightly to V-ATPase (KD 10−8 mol./l), the reversibility of the effect was attributed to de novo synthesis of the enzyme (Drose and Altendorf, 1997, J. Exp. Biology 200:1-8).
At concentrations greater than 10 nM bafilomycin A1 was reported to induce apoptosis in Capan-1 human pancreatic cancer cell line in vitro. Subcutaneous administration of doses smaller than 1 mg/kg/day did not affect the growth of nude mice for up to four weeks, and based on histological examination did not affect the liver, pancreas, small intestine, kidney or lung during the four weeks treatment. However, such treatment caused apoptotic death of Capan-1 cells in their xenograft tumor and overall shrinkage of the tumors (Ohta et al., 1998 Journal of Pathology 185:324-340).
When injected intravenously bafilomycin A1 is toxic (Keeling, 1997, Ann. N.Y. Acad Science 834:600-608). However, when administered subcutaneously bafilomycin A1 is tolerated. Doses of 1.4 mol/kg/day for 14 days were non-toxic and increased bone readsorption.
Unlike bafilomycins and concanamycins that inhibit V-ATPase from animal cells as well as from fungi and yeast, benzolactone enamides inhibit V-ATPase from animal cells alone. These compounds do not inhibit F-ATPases or P-ATPases and exert their effect on V-ATPases at the nanomolar range. Again, their inhibitory effect on the enzyme activity in vitro was exerted at lower concentrations (IC50<1 nM) than their effect on cell proliferation (IC50˜10 nM to a few hundreds nM) (Boyd and Farina et al., 2001, J. Pharmacology and Experimental Therapeutics 297:114-120).
Radiation and chemotherapy play an important role in treatment of many different types of cancers due to the preferential destruction of rapidly dividing cells by exposure to radiation or anticancer agents. However, such treatment can also destroy normal cells which reproduce rapidly such as skin, hair follicles, lining of the intestines and blood element generating components in the bone marrow. Destruction of such normal cells leads to undesirable side effects, which include nausea or vomiting, low blood cell counts (with consequent susceptibility to infections and risk of hemorrhage) and loss of hair. Therefore, improvements in radiation and chemotherapies designed to sensitize neoplastic cells or protect normal cells from the toxic effects of radiation or chemotherapy are highly desirable.